NEW PLANT BREEDING
TECHNIQUES
REPORT OF A WORKSHOP HOSTED BY FOOD
STANDARDS AUSTRALIA NEW ZEALAND
AUGUST 2013
1
DISCLAIMER
FSANZ disclaims any liability for any loss or injury directly or indirectly sustained by any person as a
result of any use of or reliance upon the content of this report.
The content of this report is a summary of discussions of an external expert panel and does not
necessarily reflect the views of FSANZ or FSANZ staff. The information in this report is provided for
information purposes only. No representation is made or warranty given as to the suitability of any of
the content for any particular purpose or to the professional qualifications of any person or company
referred to therein.
The information in this report should not be relied upon as legal advice or used as a substitute for legal
advice. You should also exercise your own skill, care and judgement before relying on this information
in any important matter and seek independent legal advice, including in relation to compliance with
relevant food legislation and the Australia New Zealand Food Standards Code.
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CONTENTS
Disclaimer .......................................................................................................................... 2
EXECUTIVE SUMMARY ................................................................................................... 4
INTRODUCTION AND BACKGROUND............................................................................. 6
DISCUSSION OF THE TECHNIQUES .............................................................................. 7
Accelerated breeding following induction of early flowering ............................................ 7
Targeted mutagenic techniques ..................................................................................... 9
Agro-infiltration for transient expression ........................................................................12
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EXECUTIVE SUMMARY
Food Standards Australia New Zealand (FSANZ) hosted a technical workshop to discuss a
number of new plant breeding techniques that have come to the attention of regulators. This
was the second workshop to be hosted by FSANZ on this topic. A number of scientists with
expertise in plant breeding and biotechnology were invited to participate in the workshop.
The objectives of the workshop were to: enhance FSANZ’s scientific knowledge and
understanding of each of the techniques; and to discuss scientific, technical and regulatory
issues, including whether derived food products should be regarded as genetically modified
(GM) food. The scientific conclusions of the workshop may constitute a relevant
consideration to which FSANZ may have regard when considering applications to amend
Standard 1.5.2 – Food produced using Gene Technology in the Australia New Zealand Food
Standards Code.
The techniques discussed were:
1.
Accelerated breeding following induction of early flowering – a technique for
shortening the flowering time in tree species, to accelerate the breeding process. It
involves using a transgenic early flowering plant line as one of the breeding parents. In
the final breeding steps, plant lines are selected that have not inherited the early
flowering transgene.
2.
Targeted mutagenic techniques – a range of techniques that have been developed for
introducing mutations at specific sites in genomes. This is in contrast to more traditional
mutagenic techniques where mutations are random. Depending on how the various
targeted techniques are deployed, mutations can either be restricted to one or a few
nucleotides or involve the insertion of a new piece of DNA. The specific techniques
discussed were:
a. transcription activator-like effector nucleases (TALENs) – artificial restriction
endonuclease enzymes generated by fusing a transcription activator-like effector
DNA binding domain to a non-specific DNA cleavage domain (nuclease).
b. type II clustered, regularly interspaced, short palindromic repeats
(CRISPR)/Cas systems – an engineered DNA targeting complex which relies on
a small guide RNA in association with an endonuclease (Cas9) to target specific
sites in the genome for cleavage.
c. meganucleases - endonucleases with large cleavage sites that can target
specific sites in the genome.
d. triplex-forming oligonucleotides - short, single-stranded, synthetic
oligonucleotides that are linked to a restriction endonuclease for targeting specific
sites in the genome.
3.
Agro-infiltration – a technique that is primarily used for the transient and localised
expression of genes in a plant typically without any integration of the introduced DNA
into the plant genome. It involves infiltrating tissues (usually intact leaves) with a liquid
suspension of Agrobacterium containing the vector. The technique was primarily
developed for use as a research tool however is now being used as a production
platform for high value proteins, e.g. vaccines.
In relation to accelerated breeding following induction of early flowering it was
concluded the final food producing lines would be comparable to those developed using a
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conventional plant breeding approach. Derived food products should therefore not be
regarded as GM food. It would be important from a safety perspective however for the early
flowering transgenic parent line to be fully characterised to make it easier to ensure any
introduced transgenes have been excluded from the final food-producing lines. This
technique is still in the research phase, therefore commercial products are not expected for
some time.
For the targeted mutagenic techniques it was concluded they are all conceptually similar to
zinc finger nuclease technology, which was discussed in the first FSANZ workshop. When
used to introduce small changes only, such techniques do not present a significantly greater
food safety concern than other forms of mutagenesis. Providing any transgenes have been
segregated away from the final food producing lines, derived foods would be similar to food
produced using traditional mutagenic techniques. Such foods should therefore not be
regarded as GM. When used to introduce a new gene however, the techniques would be
equivalent to transgenesis and, as such, any food products should be regarded as GM.
For Agro-infiltration it was concluded the technique would have limited applicability to food.
As any food products that are produced using this type of expression system will be purified
proteins, and the plants in which they are produced will not themselves be used as food,
there are no significant food safety concerns. Whether the purified protein products are
regarded as GM foods would depend on their use and whether the plants from which they
are derived are themselves GM.
Acknowledgement
FSANZ thanks all participants for generously donating their time and for enthusiastically
contributing their knowledge and expertise to the discussions.
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INTRODUCTION AND BACKGROUND
Food Standards Australia New Zealand (FSANZ) hosted a technical workshop on a number
of new plant breeding techniques. This was the second workshop to be hosted by FSANZ on
this topic. The purpose of the workshops has been to improve FSANZ’s knowledge and
understanding of various new breeding techniques and to discuss scientific, technical and
regulatory issues surrounding their potential use in commercial agriculture.
The two workshops were initiated following a number of enquiries to FSANZ regarding the
regulatory status of products generated from several new plant breeding techniques. In
contrast to the techniques used to generate genetically modified (GM) foods that have been
assessed and approved to date – which are all derived from transgenic plants – many of the
new techniques do not result in final food producing lines that are transgenic. It is therefore
not always clear whether derived food products would come within the scope of Standard
1.5.2 – Food produced using Gene Technology in the Australia New Zealand Food
Standards Code (the Code), and therefore be subject to pre-market safety assessment and
approval.
The first workshop, held in May 2012, considered the scientific question of whether foods
derived from a number of new plant breeding techniques should be regarded as GM food, or
whether they are more like conventional food. The report of the first workshop is available on
the FSANZ website (link). The second workshop considered the following additional
techniques:



accelerated breeding following early flowering;
targeted mutagenesis techniques not discussed in the first workshop; and
Agro-infiltration.
The scientific conclusions of these workshops may constitute a relevant consideration to
which FSANZ may have regard when considering applications to amend Standard 1.5.2 –
Food produced using Gene Technology.
A number of scientists with expertise in plant biotechnology and plant breeding were invited
to participate in the workshop. They were:
Name
Position
Professor Bernard Carroll
School of Chemistry & Molecular Biosciences, University of
Queensland
Dr Rob Defeyter
Intellectual Property Manager, CSIRO Plant Industry
Dr Allan Green
Deputy Chief, CSIRO Plant Industry
Dr Roger Hellens1
Science Group Leader, Genomics, Plant and Food Research NZ
Professor Peter Langridge
Director and CEO, Australian Centre for Plant Functional Genomics,
University of Adelaide
Dr Bill Taylor2
Business Development Manager, CSIRO Plant Industry
Professor Peter Waterhouse
School of Molecular Bioscience, University of Sydney
Other workshop participants were staff from FSANZ, the Office of the Gene Technology
Regulator, the Australian Government Department of Agriculture, and the New Zealand
Ministry for Primary Industries. The workshop was chaired by Professor Peter Langridge, a
FSANZ Scientific Fellow.
1
2
Dr Hellens is now Professor of Agricultural Biotechnology at the Queensland University of Technology.
Dr Taylor has since retired.
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DISCUSSION OF THE TECHNIQUES
Accelerated breeding following induction of early flowering
Overview of the technique
The main objective of this technique is to accelerate the breeding process by shortening the
time it takes for a plant to flower (juvenile stage). Some tree species can have long juvenile
stages, lasting ten years or more, which means the breeding process can be both time
consuming and costly. This is especially the case if new traits are being introduced from wild
species, where extensive backcrossing is required to eliminate unwanted traits that are
carried over in the process. Shortening the juvenile stage is therefore a very important
breeding objective for some species.
A number of different approaches exist for inducing early flowering, including transgenic as
well as more conventional approaches3. The latter have been used with varying degrees of
success but have generally not been able to reduce flowering time to less than twelve
months.
Transgenic approaches, on the other hand, have been shown to significantly shorten the
flowering time of fruit trees. They primarily involve the over-expression of various genes
involved in the flowering pathway. For example, the over-expression of flower-inducing
genes such as LEAFY (LFY), FRUITFUL (FUL), APETALA1 (AP1) or FLOWERING LOCUS
T (FT) has resulted in significant reductions in flowering times in certain fruit species. RNA
interference has also been used successfully to induce early flowering by silencing specific
genes involved in flowering repression, e.g. TERMINAL FLOWER 1-1 (TFL1-1), TERMINAL
FLOWER 1-2 (TFL1-2).
Many of the over-expressed flowering genes e.g. FT and AP1, belong to the MADS-box gene
family which all encode proteins characterised by a highly conserved DNA-binding domain
known as the MADS-box. MADS-box genes are found in animals, fungi and plants and
generally encode transcription factors.
The approach to accelerated breeding is to use the early flowering trait to facilitate the
production of a number of crossbred generations in the space of a few years. This strategy is
particularly useful for introducing single traits from distant wild species and then backcrossing
with high quality cultivars to remove any unwanted traits. Using this approach it might be
possible to achieve several backcrosses within a decade, which would be a significant
acceleration of the breeding process. In the final stages, the transgene is selected against so
that only the genes of interest (e.g. a new disease resistance gene), introduced via
conventional breeding processes, remain. The breeding process therefore commences with
a transgenic plant as one of the parental lines but the final food producing lines will not be
transgenic.
One of the best known examples of using this approach is the work by Flachowsky et al.
(2011)4 with transgenic apples over-expressing the BpMADS4 gene from silver birch. The
BpMADS4 gene is a homologue of the FUL gene from A. thaliana and a member of the
MADS-box gene family. A single transgenic line was selected which flowered within a few
months. This line is being used in a cross breeding programme to introgress fire blight
3E.g.
selection of naturally precocious breeding stocks, root pruning and girdling, grafting onto specialised
rootstocks, application of growth regulators, imposition of stress, and intensive management of plant nutrition.
4 Flachowsky H, Le Roux P-M, Peil A, Patocchi A, Richter K, Hanke M-V (2011) Application of a high-speed
breeding technology to apple (Malus x domestica) based on transgenic early flowering plants and marker-assisted
selection. New Phytologist 192:364-377
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resistance from wild apple (Malus fusca) and combine that trait with several resistance genes
to apple scab and powdery mildew. Transgenic seedlings carrying the combined resistance
traits will then be crossed with ‘Golden Delicious’ to continue elimination of unwanted traits
acquired from the wild species. During the backcrossing process with other elite cultivars
non-transgenic, multi-resistant seedlings can be selected which can be further used in a
classic breeding programme to obtain the final commercial lines. While this remains a
lengthy and complex process, it is nevertheless significantly faster than the classic plant
breeding approach.
While this technique is mainly being exploited for tree breeding, broader applications are also
being considered. For example, in temperate cereals to convert winter to spring genotypes
as a way of allowing multiple generations per year.
Discussion
The main points from the discussion are summarised below:

In terms of any changes, both intended and unintended, arising from the inserted
transgene and the early flowering phenotype, these will be confined to the early
generations as the process results in the transgene being segregated away during the
latter stages of the breeding process.

The most important thing from a safety perspective would be for the starting transgenic
line to be fully characterised so that the number of copies of the transgene in the original
event are known. It would then be reasonably straightforward to ensure all insertions
have been excluded from the final food-producing lines.

Once any introduced transgenes have been segregated away, any changes associated
with those transgenes should no longer be present in the final food producing lines or
products. The final food producing lines and derived food products would therefore be
comparable to those derived using a conventional plant breeding approach.

While this technique would be successful in accelerating the time it takes to do the initial
crosses, a significant amount of time would still be required before an acceptable
commercial product might be developed. Commercial products are therefore not
expected for some time.

Some parallels exist between the early flowering technique and some of the other
techniques discussed in the first workshop such as seed production technology and
reverse breeding. In all three cases, a transgenic plant line is used in the early stages to
facilitate the breeding process, but the final food-producing lines are non-GM.
Conclusion
Providing the breeding process results in complete removal of the early flowering
transgenes, the final food producing lines will not be transgenic. Food products derived using
this technique should therefore not be regarded as GM food.
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Targeted mutagenic techniques
Overview of the techniques
The techniques discussed were:




transcription activator-like effector nucleases (TALENs)
type II clustered, regularly interspaced, short palindromic repeats (CRISPR)/Cas
systems
meganucleases
triple helix-forming oligonucleotides (TFO)
These techniques described in Table 1 below, which also includes zinc finger nuclease
(ZFN) technology discussed in the first FSANZ workshop, can all be regarded as different
tools for achieving the same objective – the introduction of a double stranded break (DSB) at
a specific site in the plant genome. Once the DSB has been made, the options for
introducing a mutation at the break site are the same irrespective of the technique used.
Mutations are introduced using the cells own repair pathways - either non-homologous end
joining (NHEJ) or homology-directed repair (HDR). The two repair pathways differ in their
fidelity and template requirements, with NHEJ being the dominant repair pathway in plants.
In NHEJ, the cleaved ends are modified then directly ligated using little or no sequence
homology. In HDR, a homologous sequence is required to guide the repair. In normal
circumstances, a homologous repair template is provided by the sister chromatid, leading to
the faithful restoration of the original sequence. However, to introduce a specific change at
the break site, an exogenous DNA template can be provided. The resultant changes can
range from point mutations to the insertion of new genes.
In terms of delivery to the plant cell, techniques such as ZFN technology, TALENs and
CRISPR/Cas9 typically use either Agrobacterium-mediated or protoplast transformation.
Where this results in stable, rather than transient, gene expression, the intention is to
segregate out the introduced DNA prior to commercialisation. In the case of the
meganucleases, because of their stability, these can be directly transferred to the plant cell
(e.g. by biolistics or electroporation) or indirectly transferred via mRNA which is then
translated in the plant cell. Both delivery methods circumvent the introduction of DNA and its
subsequent integration into the host genome. In the case of TFOs, delivery is not possible
via Agrobacterium transformation and hence relies on techniques such as biolistics,
electroporation, polyethylene glycol-mediated transformation and silicon carbide whiskersmediated transformation.
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Table 1: Description of the various targeted mutagenic techniques
Technique
DNA recognition and cleavage
Comments
Zinc finger
nuclease (ZFN)
technology5
DNA recognition is by an engineered array of zinc finger
DNA binding domains, each interacting with three
nucleotides. Cleavage is by a non-specific endonuclease
domain derived from Fok1.
ZFN dimers can target up to 36-bp sequences. Adjusting the specificity of ZFNs
relies on the shuffling of domains with established triplet specificity.
Transcription
activator-like
effector nucleases
(TALENs)
DNA recognition is by an engineered transcription activatorlike effector (TALE) protein consisting of an array of 12 to
26 repeats, each interacting with a single nucleotide. Like
ZFN technology, cleavage is by a non-specific
endonuclease domain derived from Fok1.
The construction of engineered TALENs is challenging but a variety of assembly
methods have been developed, including automatable high-throughput
techniques. TALENs have been used for targeted mutagenesis in Arabidopsis,
tobacco, rice and Brachypodium (bunch grasses) and are predicted to be
extended in the near future to crops such as soybean, potato and canola.
DNA recognition is by an artificial single guide RNA
(sgRNA) that has embedded within it a sequence
complementary to the target sequence. Cleavage is by a
type II CRISPR-associated nuclease (Cas9).
The RNA-guided Cas9 can function in a variety of cells and organisms to
introduce DSBs at specific sites. Because the CRISPR/Cas9 system relies on
duplex or triplex formation between RNA and DNA it potentially has much higher
specificity than protein-based systems. Only a short fragment in the sgRNA
needs to be designed to target the desired sequence in contrast to ZFN
technology or TALENs where complex manipulations of proteins are required.
CRISPR/Cas systems have so far been used for targeted mutagenesis in
Arabidopsis, tobacco, sorghum and rice.
Clustered, regularly
interspaced, short
palindromic repeats
(CRISPR)/CRISPRassociated (Cas)
system
Meganucleases
Triplex-forming
oligonucleotides
(TFOs)
5
6
DNA recognition is by a DNA binding domain which binds
to very long (12 – 40 bp) and sometimes asymmetric
recognition sequences. In meganucleases, the DNA
binding domain is not clearly separated from the catalytic
(cleavage) domain. This makes meganucleases difficult to
engineer for targeting specific DNA sequences.
DNA recognition is by short, triple-helix forming synthetic
oligonucleotides which are conjugated to a restriction
endonuclease.
Re-designed meganucleases with tailored substrate specificity have the
advantage of being extremely specific as well as very stable proteins. A redesigned meganuclease from Chlamydomonas reinhardti chloroplast(1-CreI)has
been used to successfully introduce mutations into the liguleless1 locus of maize
(Gao et al 2010)6.
Application of this approach is mainly hampered by the restriction of triple-helix
formation to DNA fragments with strands composed of either purines or
pyrimidines. The overall efficiency of the TFO technique is low but the high
specificity (as a result of nucleic acid-DNA recognition) ensures off-target effects
are minimal.
ZFN was discussed in detail at the first FSANZ workshop on New Plant Breeding Techniques and is included here for comparison.
Gao et al (2010) Heritable targeted mutagenesis in maize using a designed endonuclease. The Plant Journal 61: 176-187
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Discussion
The main points from the discussion are summarised below:

Most interest is centred on developing the ZFN, TALENS and CRISPR/Cas9
systems. There is little interest in pursuing the meganuclease or TFO techniques for
targeted mutagenesis in plants.

One of the main issues to consider in relation to the various techniques is their
specificity, in particular the possibility of unintended (off-target) effects. In general, the
longer the recognition site in the DNA, the greater the specificity. In the case of ZFN
technology however a long recognition site would require the engineering of a very
large ZFN, which would be quite challenging. Whereas for TALENS, the addition of
extra repeats to accommodate a longer recognition site would probably be much
easier. The main problem with recognition by protein/DNA binding is that the current
understanding of the interaction is still quite limited. In contrast, knowledge of how
nucleic acids bind is well developed and the rules for interaction are quite precise. A
RNA/DNA interaction is probably more rigorous than a DNA/DNA interaction.
Because it relies on nucleic acid binding, rather than protein-DNA binding, for
recognition, the CRISPR/Cas9 system will have greater specificity compared to the
ZFN and TALENS systems.

Stable transformation remains the preferred method for delivering the nuclease
constructs to the cell, although transient delivery systems are also being investigated.
Where stable transformation is used, the aim is to segregate away the introduced
construct once the desired mutation has been obtained so that no transgene remains
in the final food producing line.

While both NHEJ and HDR are the main repair mechanisms for DSBs in cells, the
occurrence of natural HDR in plant cells is actually very rare (possibly one in a
million). It is likely, therefore, that even when a template is provided to direct repair of
a DSB, the repair mechanism used by the plant cell will be NHEJ, rather than HDR.
NHEJ repair is typically accurate and efficient but is prone to the formation of
sporadic unfaithful repair products, such as small deletions or insertions, frequently
leading to gene disruption.

The same general principles and issues which were considered in the first workshop
in relation to foods derived using the various ZFN techniques also apply to these
other targeted mutagenic techniques. It was noted that the specificity of the
techniques is improving all the time which should further limit any potential for offtarget effects.
Conclusion
The mutagenic techniques discussed at this second workshop are conceptually similar to
ZFN technology. When used to introduce small changes, such techniques do not present a
significantly greater food safety concern than other forms of mutagenesis. Providing the
introduced DNA has been segregated away from the final food producing lines, food derived
from plants modified using these techniques would be similar to food produced using
traditional mutagenic techniques, and should therefore not be regarded as GM food. When
used to introduce a new gene however, the techniques would be equivalent to transgenesis
and, as such, any food products should be regarded as GM.
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Agro-infiltration for transient expression
Overview of the technique
Agro-infiltration is a technique that was primarily developed as a research tool for evaluating
the activity and function of candidate genes and promoters without the need to stably
transform plants. The technique involves cloning the genes/promoters of interest into a
specific vector which is then transformed into Agrobacterium. The Agrobacterium, in a liquid
suspension is infiltrated into the intercellular space of the plant tissue where it transfers the
gene of interest to the plant cell. Expression is usually only transient and occurs without any
integration of the introduced DNA into the plant genome because the target cells are usually
not dividing. That is, the plants are not transgenic plants. Agro-infiltration can however also
be used to develop transgenic plants if, for example, flowers that contain germ line cells are
the target tissue for infiltration. Agro-infiltration can be performed with a variety of expression
vectors, including non-viral or plant virus-based vectors.
The most common tissue that is used as a target for infiltration is leaf. A suspension of the
bacterium can be infiltrated into the leaf of an intact plant, or alternatively leaf discs, leaves or
whole plants can be submerged in the bacterial suspension under vacuum. The technique
has been used successfully for a variety of plants including tobacco, Arabidopsis, grape, pea
and flax.
While primarily used as a research tool, the technique is also being developed as a
production platform for high value products such as pharmaceutical proteins (e.g. vaccines),
which are normally produced in cell culture. This type of technology could also potentially be
used to produce proteins, particularly enzymes, used in food processing.
The key features of Agro-infiltration as a transient expression system are:

plant material is destroyed or harvested at the end of the production process

production is almost always contained within a glasshouse or laboratory

it is rapid, scaleable with very high expression levels being achieved

it is relatively inexpensive and easy to use
Discussion
There was considerable discussion around whether the system would actually be used for
food substances that are low in value compared to pharmaceutical products. It was noted
that in the area of enzyme production, bacterial fermentation systems are already very costeffective. It was also suggested that stable transformation would be a more efficient system
than transient expression for industrial-scale production of food substances.
The key points from the discussion were:

From the food perspective, the most likely substances to be produced will be food
processing enzymes or food additives and potentially also protein supplements. It
was noted that food processing enzymes and food additives are not regarded as
food, and are regulated under separate Standards in the Code, irrespective of
whether or not they have been produced using GM techniques.

For food products such as protein supplements, whether they are regarded as GM
food would depend on whether the expression vector becomes stably integrated into
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the plant genome. Such integration events may occur at low frequency in the
infiltrated area, however if only somatic (non-germ line) cells are involved the
integrated DNA will not be inherited in the next generation.

As the food products that are produced using this type of expression system will be
purified proteins, and the plants in which they are produced will not be used as food,
the technique does not raise a potential food safety concern.
Conclusion
The technique is considered to have low applicability to food although it could be envisaged
as a potential production platform for proteins to be used as food. In this case, it does not
raise any food safety concerns. Whether any purified protein products are regarded as GM
foods would depend on their use and whether the plants from which they are derived are
themselves GM.
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